Mathematical Prediction of Cooling Rates during Cryopreservation of Reproductive Cells in Liquid Nitrogen

Abstract

Liquid nitrogen is one of the cryogenic fluids most used for cryopreservation of animal germoplasm; low temperature preservation of oocytes, sperm and embryos is a fundamental cornerstone of assisted reproductive technologies. The cryopreservation protocol for sperm is important because cooling rates that are too high or too low can be detrimental for reproductive cells. A widespread practice is to freeze plastic straws containing sperm in static nitrogen vapor over liquid nitrogen for variable periods of time before plunging into liquid nitrogen for indefinite storage. Regarding oocytes, the cryopreservation of this gamete has increased in recent years due to the application of in vitro fertilization, nuclear transfer and the need to establish ova/gene banks worldwide. Current advances in cryopreservation of oocytes indicate that high cooling methods (vitrification) achieve the best results since there is a minimum risk of intra/extra-celullar ice-formation. Vitrification is a process in which liquid water is converted into a glass-like amorphous solid without any ice formation; it can be achieved by an extremely rapid cooling of a small sample and/or the introduction of cryoprotectant agents that suppress the formation of ice crystals. In order to minimize the working volumes of solutions, several minimal volume devices have been commercially introduced in the last years, such as open pulled straws (OPS), fine and ultra fine pipette tips, nylon loops and polyethylene films. The usual practice is to plunge the devices containing biological samples directly into the liquid nitrogen.